Infectious disease remains the leading cause of mortality worldwide. A significant aspect of this problem is the continuing rise of infections that are resistant to most, if not all, conventional antibiotics. To meet this challenge it is essential that new drug targets be identified, and new classes of antibiotics developed. Over the past two decades a large number of naturally-occurring antimicrobial peptides have been found in both vertebrate and invertebrate species that are capable of providing a rapid and broad-spectrum response against a wide variety of pathogens. Because the specificity of these peptides is based on recognition of general properties of the cell membrane the emergence of resistance is exceedingly rare, making them ideal starting points for the development of new antibiotics. A limiting factor in our ability to further enhance the efficacy of these peptides is the lack of detailed knowledge about their mechanism of action, and in particular the manner in which they interact with and disrupt the cell membrane. The goal of this proposal is to develop a clear understanding of peptide-membrane interactions and mechanism of action for a promising and well-defined class of antimicrobial peptides that are synthetic hybrids of the insect peptide cecropin A and the bee-venom peptide, mellitin. Site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy provides a powerful and well-established approach for the analysis of peptide-membrane interactions that is uniquely suited to providing such a detailed understanding. Specifically, we will use both conventional and pulsed SDSL EPR to measure membrane binding affinities, determine structure, topology, degree of membrane insertion, and peptide-peptide interactions for cecropin-mellitin hybrid peptides in model membranes that mimic both eukaryotic and bacterial membranes and in intact cells. These properties will be related to antibiotic efficacy against a panel of drug-sensitive and drug-resistant bacteria. We will systematically modify peptide composition to define relationships between sequence, membrane interactions, and antibacterial efficacy. Finally, we will synthesize and evaluate the antibiotic efficacy and membrane interactions of peptidomimetic analogs composed of non-natural beta-amino acids. These studies will significantly advance our understanding of the mechanism of action of antimicrobial peptides, and contribute to the further development of peptide and peptidomimetic antibiotics.